Stepped cannula

ABSTRACT

Described herein are cannulas having a stepped exterior. Also described are methods of making and using these cannulas, for example to deliver one or more materials to the central nervous system of an animal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/243,756, filed Oct. 5, 2005, which claims the benefit of U.S.provisional applications 60/616,238, filed Oct. 5, 2004 and 60/641,551,filed Jan. 4, 2005, all of which applications are incorporated byreference herein in their entireties.

TECHNICAL FIELD

This invention is in the field of cannulas. In particular, the inventionrelates to cannulas for delivering a material, for example abiologically active agent, into the central nervous system, to systemscomprising these cannulas as well as to methods of making and usingthese cannulas.

BACKGROUND

Cannulas can be used to deliver materials into the central nervoussystem (CNS) of a subject. However, with current cannula designs, caremust be taken to prevent reflux of the material along the injectiontrack. Quereshi et al. (2000) Neurosurgery 46(3): 663-69. Even withprecautions are taken to minimize reflux, such as slow removal of thecannula and the application of pressure to the tissue as the cannula isremoved, reflux remains a problem.

In addition, a substantial portion of the material being delivered canbe lost due to exposure of the material to the large surface area of theinside of the cannula. In particular, exposure to stainless steel cancause substantial loss of the material to be delivered. For example,various groups have demonstrated that a substantial amount of adenovirusvectors preparations exposed to stainless steel surfaces are lost.Naimark et al. (2003) Hum. Gene Ther. 14:161-6; Tsui et al. (2001) Mol.Ther. 3:122-5; Marshall et al. (2000) Mol. Ther. 1(5 Pt 1):423-9. Theproblem is exacerbated when very small volumes of material are beingdelivered, because the smaller the volume, the greater the ratio ofsurface area to volume within the cannula. Given that the use of smallvolumes of material is particularly desirable in situations where thematerial is expensive or difficult to obtain, it would be desirable tohave devices and methods in which both reflux and loss of material areminimized.

Thus, there exists a need for a cannula capable of introducing materialsinto the brain of a subject without reflux of the material along theneedle track. A need also exists for cannula designs that reduce theloss of agents to the inner surface(s), and, accordingly, can deliversmall volumes of material effectively.

SUMMARY

The present invention solves these and other problems by providingcannula designs that reduce or eliminate reflux and/or loss of thedelivered material.

In one aspect, the present invention relates to cannulas for thedelivery of agents to a target tissue in an animal. In some embodimentsthe target tissue is the central nervous system (e.g., brain). In someembodiments the agent is a biologically active agent.

In one embodiment, the cannula comprises an external step design inwhich the diameter of the cannula in contact with the material to bedelivered decreases in a stepwise fashion at defined points along itslength. Thus, in one aspect, the invention includes a stepped cannulahaving an exterior diameter, a distal end, a proximal end and a lumenextending between the proximal and distal ends, the stepped cannulacomprising two or more co-axially disposed segments, each segment havingan exterior diameter that defines the exterior diameter of the cannula,wherein the exterior diameter of the segments is different.

In certain embodiments, the exterior diameter has the step configurationwhile the interior surface in contact with the material does not havethe step configuration.

In any of the embodiments described herein, the decrease in diameter maybe in a proximal to distal direction (i.e., the step at the proximal endof the cannula having the largest diameter and the step at the distalend having the smallest diameter). There may be any number of stepsalong the exterior diameter, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or even more.

In any of the embodiments described herein, the diameter of the stepsmay increase by the same amount from step to step (i.e., the differencein diameter between adjacent steps is uniform). Alternatively, in any ofthe embodiments described herein, the difference in diameter of thesteps may vary from step to step along the length of the cannula. Inaddition, in any of the embodiments described herein, the distancebetween steps may be the same or it may vary. In one embodiment, thecannula has the structure and dimensions as described below in Example 1and in reference to FIGS. 3A and/or 3B.

The cannulas described herein may be made of any material, includingmetals, metal alloys, polymers or combinations thereof. In certainembodiments, the cannula comprises stainless steel exterior with anon-stainless steel surface that contacts the product to be delivered.For example, the surface of the lumen of the cannula (which is contactwith the material to be delivered) may be comprised of a polymericcoating over the stainless steel. Alternatively, the cannula may furthercomprise one or more tubes extending through the lumen of the cannula,for example fused silica tubing encased in the stainless steel outersleeve of the cannula, such that in use product contacts the innersurface of the fused silica tubing rather than stainless steel. As notedabove, the surface in contact with the material to be delivered may ormay not have a step configuration. In yet another embodiment, thecannula is constructed as shown in FIGS. 3A and 3B from the materialsdiscussed below.

In any of the cannulas described herein, the cannula may comprise two ormore materials. In certain embodiments, a stainless steel cannulasurrounds a fused silica tubing, in which the surface contacted by thematerial to be delivered is quartz silica. In other embodiments, astainless steel exterior surrounds a fused silica inner surface, inwhich the surface contacted by the material to be delivered is fusedsilica. In a preferred embodiment the cannula has the structure,dimensions and is made of the materials as described in Example 1 andshown in FIGS. 3A and 3B.

In another aspect, the invention includes a cannula assembly comprising:any of the cannulas described herein and a reservoir comprising the oneor more materials to be delivered through the cannula, the reservoiroperably connected to the lumen of the cannula. The one or morematerials (e.g., potentially therapeutic formulations), are alsoreferred to herein as “product(s)”. In certain embodiments, thereservoir comprises a syringe. Further, in any of the systems orassemblies described herein, the cannula and/or reservoir can beoperably linked to one or more pumps (e.g., syringe pumps). In certainembodiments, the cannulas are operably linked to the pump(s) via tubingthat extends through the lumen of the cannula. In certain embodiments,the systems described herein further comprise a stereotactic frame (see,e.g., FIG. 5).

The materials delivered by these systems may comprise one or morebiologically active agents (e.g., AAV vectors, proteins, drugs, etc.),dyes, tracers, markers, contrast agents or combinations thereof.Furthermore, the systems may be used for delivery to any part of thebody, most preferably to the brain of an animal. In one embodiment, theinvention provides a cannula that has a decreased hold-up volume.

In another aspect, the invention includes a method of delivering one ormore materials to a target area in a subject, the method comprising thesteps of positioning a cannula or cannula assembly as described hereinat the target area of the subject; and delivering the one or morematerials to the target area through the cannula. In certainembodiments, the target area is in the central nervous system, forexample, the brain.

These and other embodiments of the subject invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an overview of an exemplary system comprising a steppedcannula 2 as described herein. Also shown is a syringe 4, which islinked to the cannula 2 via fused silica tubing 1, 3 inside FEP Class VItubing 3, and a syringe pump 5. Syringe pump 5 is connected to tubing 3via Luer compression fitting 6.

FIG. 2 is a side view of another exemplary system comprising anexemplary stepped cannula 10 as described herein. Also shown is asyringe pump 16 and a syringe 15, which is linked to the cannula 10 at aLuer compression fitting 14 via fused silica tubing 12 extending throughthe lumen of a FEP (Teflon) tubing 13 and the cannula 10.

FIG. 3A depicts side views showing exemplary steps involved in theassembly of an exemplary injection needle sub-assembly (INSA) asdescribed herein.

FIG. 3B-1 through 3B-6, are side views depicting various steps involvedin the assembly of an exemplary injection needle assembly (INA) asdescribed herein.

FIG. 4 depicts an overview of an exemplary Medfusion 2010i syringe pumpwith an attached syringe.

FIG. 5 is an overview of an exemplary cannula as described hereinattached to a stereotactic frame for administration of product to ahuman subject.

FIG. 6 is an overview of another exemplary cannula as described herein.

FIG. 7, panels A-D, depict results of immunohistochemical staining foraromatic L-amino acid decarboxylase (AADC) in whole mounted brainsections from monkeys used in the experiments described in Example 2.Brains are shown in coronal section through the infusion site at 5.5weeks post-infusion. Panels A, B, C, and D represent the four differentmonkey brains analyzed in Example 2 [MR15102M (A), MR15109M (B), R23700M(C) and R211101M (D)]. All left hemispheres received ramped infusion andall right hemispheres received non-ramped infusion. The black arrowindicates the putamen region.

FIG. 8, panels A and B, depict high magnification images ofimmunohistochemical staining for AADC within the putamen of the brain ofa monkey used in the experiments described in Example 2 at 5.5 weekspost-infusion. A representative section from the hemisphere thatreceived ramped infusion is shown in FIG. 8A and one from the hemispherethat received non-ramped infusion is shown in FIG. 8B. The scale barrepresents 100 μm.

FIG. 9, panels A and B, depict Hematoxylin and Eosin (H&E) stainedsections within the putamen from a representative animal used in theexperiments in Example 2, R211101M, at 5× magnification. Animal R211101Mreceived bilateral CED of AAV-hAADC-2 using the non-ramped infusionprocedure in the right hemisphere (FIG. 9A) and the ramped infusionprocedure in the left hemisphere (FIG. 9B). Images illustrate the areaadjacent to the cannula track, and were taken at the mid-caudal putamenlevel. The scale bar represents 400 μm.

DETAILED DESCRIPTION

The present invention relates to novel cannulas for delivery ofmaterials (e.g. formulations comprising potentially therapeutic agents)to a target tissue of an animal, such as the brain. The cannulasdescribed herein greatly reduce or eliminate reflux during delivery ofthe materials. Such materials are referred to herein generally as“product.” More specifically, the present invention enables delivery ofproduct to well-defined locations within the brain of a subject withminimal reflux of product along the needle track, with minimal hold-upvolume, and with minimal losses of product to the internal surfaces ofthe cannula.

In one embodiment of the present invention, the cannula has a stepdesign in which the diameter of the cannula decreases in a stepwisefashion at defined points along its length (from proximal to distalregion). Thus, in preferred embodiments, the smallest cannula diameteris at the distal most portion of the cannula. As noted above, this stepdesign reduces reflux of product along the needle track. In oneembodiment the exterior surface of the cannula comprises five segmentsdiffering in external diameter, forming four steps, and in anotherembodiment it has the structure and dimensions discussed below. Thesurface of the cannula may be smooth, as in the embodiment illustratedin FIGS. 3A and 3B.

FIG. 1 shows an overview of an exemplary system comprising a steppedcannula 2 with a stainless steel exterior. Fused silica tubing 1, 3extends through the lumen of cannula 2 and links cannula 2 to syringe 4via hub and/or blunt needle 6 on the syringe 4. Syringe 4 is alsoattached to computerized syringe pump 5. The cannula includes a meansfor reducing or eliminating reflux of the material to be delivered, forexample, tubing (e.g., fused silica) which extends through the lumen ofthe stepped cannula and is in contact the material to be delivered.

The exemplary embodiment depicted in FIG. 1 shows a cannula 2 having atotal of four “steps.” It will be apparent that the steps nearest thedistal end of the cannula are those that enter the target tissue first,and, accordingly, the number of steps entering the target tissue e.g.brain) will depend on the depth of penetration needed to reach thattarget in the subject animal. With respect to delivery to the brain, theoperator can readily determine the appropriate depth of penetration,taking into account both the size of the animal being treated and thelocation within the brain that is being targeted.

As shown in FIG. 1 the exterior diameter of the cannula 2 decreases ateach step along the length of the cannula, in a proximal to distaldirection. As used herein, proximal refers to points close to thesyringe 4 from which product is dispensed, and distal refers to pointsclose to the point of ultimate product delivery (e.g. the targettissue).

FIG. 1 depicts an exemplary embodiment in which the two proximal-mostsegments, which border the proximal-most step, have approximately thesame length, while the four distal-most segments are of varying lengthsfrom each other and from the two proximal-most segments. Thus, it willbe apparent that some, all or none of the segments between the steps mayhave the same length as other segments.

Non-limiting examples of materials which may be used to the variouscomponents of the cannula and/or systems comprising the cannula areshown in the following table:

Component (in reference to FIG. 1) Part Source Composition ProductContact Tubing 1 at Fused Silica Polymicro Quartz silica and Yes; Silicaportion distal end of Tubing at tip Polyimide coating only cannulaCannula 2 23 G to 15 G Ranfac Stainless Steel No steel tubing Tubing 3*Fused Silica Polymicro Quartz silica and Yes; Silica portion connectingTubing Polyimide coating only cannula 2 to syringe 4 Syringe 4 SyringeBD USP Class VII Yes Polypropylene Pump 5 Pump Medfusion Multiplematerials No Luer joint 6 Luer Hub/blunt BD (USP Class VII Yes needle(from 23 G × Polypropylene) and 1½ needle) Stainless Joints** Gluejoints Locktight Cyanoacrylate Yes *fused silica inside FEP Class VItubing. FEP tubing does not have product contact **between 1 and 2;Between 2 and 3; Between 3 and 6

FIG. 2 shows an overview of an exemplary system similar to that shown inFIG. 1. The embodiment shown in FIG. 2 comprises a stepped stainlesssteel cannula 10 with fused silica tubing 12 running through the lumenof the stainless steel cannula and extending beyond the distal end ofthe cannula 10. Also shown in FIG. 2 are tubing (FEP) 13 covering thefused silica tubing 12, as well as a Luer compression fitting 14 and 1inch stainless steel (23 ga) between fused silica tubing 12 and FEPtubing 13. The Luer compression fitting 14 is connected to a syringe 15,which in turn is connected to a pump 16.

Exemplary materials and exemplary commercial sources of these materialsthat can be used in making an embodiment such as that shown in FIG. 2are set forth in the following table:

Component Exemplary (reference Commercial Product # in FIG. 2) SourceComposition Contact Cannula 10 Ranfac 304 SS No Fused Silica PolymicroFused silica Yes Tubing 12 Technologies w/polyimide coating on outsideTeflon Tubing 13 Western Analytical Teflon ® No Products (FEP) Luerfitting 14 Upchurch Scientific Polypropylene Yes with ETFE Syringe 15 BDPolypropylene Yes Pump 16 Medfusion N/A No

As shown, in certain embodiments, tubing extends through the lumen ofcannula and the product(s) to be delivered are delivered through thistubing. In embodiments containing the tubing, the tubing may be flushwith the distal end of the cannula. Alternatively, in preferredembodiments, the tubing extends from the distal end of the cannula. Insuch embodiments, the amount which the tubing extends may vary dependingon the application. Generally, the tubing will extend from about 1 mm toabout 1 cm from the cannula (or any length therebetween), morepreferably from about 1 to about 50 mm (or any length therebetween), andeven more preferably from about 1 mm to about 25 mm (or any lengththerebetween, including, but not limited to, 1 mm, 2 mm, 3 mm, 4 mm, 5mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm or 25 mm). Inone preferred embodiment, the tubing extends approximately 10 mm beyondthe distal end thereof.

As shown in the Figures, the tubing extending through the cannula mayhave one more coatings or surrounding materials in one or more regions,for example to protect the tubing in contact with the produce to bedelivered. Thus, in certain embodiments, tubing (e.g., FEP (Teflon)tubing) protects the portion of the fused silica tubing extending beyondthe proximal end of the stainless steel cannula. The fused silica tubingmay be connected to the syringe by any suitable means, including, butnot limited to, a Luer compression fitting, and the syringe is driven bya syringe pump (manual, electronic and/or computerized). It willapparent that the syringe size can be selected by the operator todeliver the appropriate amount of product(s). Thus, 1 mL, 2.5 mL, 5 mL,or even larger syringes may be used.

In certain embodiments, the Luer compression fitting comprises a 1 inchstainless steel 23G spacer between the fused silica (inner) tubing andthe FEP (outer) tubing. The optional spacer, provides mechanicalrigidity at the Luer compression fitting and helps seal the gap betweenthe inner and outer tubing when the ferrule is glued in place usingLoctite® adhesive. This gap must be filled to prevent product fromentering the space between the inner and outer tubing as it is beingadministered to a subject. Preferably, the proximal end of the spacerrepresents the only stainless steel product contact surface of thesystems and cannulas described herein. This minimal stainless steelproduct contact surface may be eliminated if desired by applyingLoctite® adhesive or other coating to cover the otherwise exposed end ofthe spacer, to provide a system with absolutely no stainless steelcontact with product. Alternatively the spacer could be comprised of adifferent material.

FIG. 3A depicts selected exemplary steps in making a stepped cannula asdescribed herein. See, also, Example 1. In particular, the step designcannula that reduced reflux may be assembled in the order shown by thearrows (top to bottom), namely by adding components (20, 22, 24, 26, 28,30) of increasing diameters. Thus, as described in Example 1, variouslength segments are joined together to form the step design.

When the cannula is made from two or more pieces, the joints should notallow materials to leak from the cannula into the target tissue or viceversa (from the target tissue into the cannula). Accordingly, the jointsare preferably sealed. The joints can be sealed in a variety of ways,including but not limited to, welding (e.g., laser welding), adhesives,sealants, heating (e.g., for thermoplastic polymers) and combinationsthereof. It will be apparent that the nature of the seal will depend onthe material used to make the cannula, for example welding may be usedfor stainless steel cannulas while heating may be used for thermoplasticpolymers.

A step design cannula as described herein can also be formed in a singleintegral piece, for example by injection molding a stepped cannula asdescribed herein.

FIGS. 3B1-6 depict assembly of an exemplary stepped cannula as describedherein. As described in Example 1, the stepped cannula 35 shown in FIG.3A is prepared by removing the needle guard 32 and inserting an innertubing component 40 through the cannula 35 until it extends from theends of the cannula. (FIG. 3B-1). Any material may be used for the innertubing component 40, including but not limited to fused silica tubing.

As an alternative to tubing, it will be apparent that the inside of thesteel cannula 35 can be coated with one or more materials that contactthe product to be delivered, thereby reducing loss of the product to thesteel cannula during delivery. Various techniques of coating ofstainless steel materials are known and may be used.

Optionally, adhesive may be placed on the tubing 40 such that the tubingis secured to the needle. Any suitable adhesive can be used, forexample, Loctite® adhesive. Preferably, the bond strength of theadhesive is at least about 4 lbs, more preferably at least about 5 lbs.

In the embodiment shown in FIG. 3B, the needle guard 32 may be replacedand a previously-cut length of tubing 31 (e.g., FEP tubing) extendedover the fused silica tubing 40 through the cannula 35 (FIG. 3B-2). Thelength of the outer tubing 31 can be determined by the indication andcan range from 10 inches to 5 yards in length (or any valuetherebetween). Thus, in certain embodiments, the outer tubing covers thefull-length of the inner tubing and may extend over the inner tubing.Alternatively, in other embodiments, the outer tubing 31 does not fullyextend over the length of the inner tubing 40 (FIG. 3B-2). Any suitableadhesive may be used to secure the outer tubing 31 to the assembly, forexample at the ends of the outer tubing 31. The bond strength of theadhesive is preferably at least about 5 lbs.

As shown in FIG. 3B-3, one or more spacer components 47 may be insertedover the inner and/or outer tubing 40, 31. The spacer 47 may be made ofany material including metals, metal alloys, polymers and combinationsthereof. In certain embodiments, the spacer 47 comprises stainlesssteel. The spacer 47 can be any length, although it is preferably thatdoes not extend over the needle. Optionally, a component may be includedto help seal the components of the assembly, for example a length of PVCshrink tubing 49. See, Example 1 for exemplary dimensions of space andPVC tubing components.

Subsequently, the assembly may be fitted with one or more componentsthat allow it to be conveniently linked to a product delivery reservoir.For example, as shown in FIG. 3B-4, appropriately sized female Luercompression fitting 50 is slid over a length of the outer tubing 31 anda ferrule 51 is placed over the outer tubing 31, preferably such that itis flush with the end of the outer tubing 31. Adhesive may be optionallyapplied to one or more of the components (e.g., outside of the end ofouter tubing prior to fitting of ferrule on the end and/or to seal thejoints between the inner tubing, spacer, outer tubing and ferrule.

The length of inner tubing 40 extending from the ferrule 51 may beremoved, for example by scoring the tubing and snapping or cutting itoff and the ferrule 51 fitted inside of the Luer compression fitting 50(FIG. 3B-5). The shrink tubing 49 may be heated to seal the joint.Finally, a male Luer compression fitting 55 can be assembled and fittingonto the female Luer compression fitting 50 and ferrule 51.

As noted above, the stepped cannulas described herein may be made out ofthe variety of materials that are physiologically acceptable, includingbut not limited to metals, metal alloys, polymers, organic fibers,inorganic fibers and/or combinations thereof. In preferred embodiments,the cannula comprises stainless steel (e.g. 316SS or 304SS).

Optionally, a product-contact surface (e.g., tubing or coating) mayextend through the lumen of the cannula. A variety of materials may alsobe used for the optional product-contact surface, including but notlimited to metals, metal alloys, polymers, organic fibers, inorganicfibers and/or combinations thereof. Preferably, the product-contactsurface is not stainless steel. In such embodiments, the outer cannulamust still be made of a material physiologically compatible with thetarget tissue, but there since there is no product contact it need notbe compatible with the biologically active agent or product formulation.Similarly, in such embodiments the FEP (Teflon) tubing shown in theFigures may be replaced with other tubing without regard to whether thetubing material is compatible with the biologically active agent orproduct formulation.

Thus, in one embodiment, the product-contact surface of the cannulacomprises or consists of fused silica (e.g., quartz silica and polyimidecoating) (Polymicro, Phoenix, Ariz.). The use of fused silica for theproduct contact surfaces greatly reduces losses of product when comparedwith prior art cannulas, in which product is exposed to stainless steel.Indeed, while only 59±14% of an adeno-associated virus vector wasrecovered from a prior art injection device that had been pre-flushedwith product, 101±6% was recovered from a device comprising a cannula ofthe present invention even without pre-flushing. See, Example 2.

One of skill in the art would realize that materials other than fusedsilica may be used in cannulas of the present invention, provided thatsuch materials have the property of low surface-related losses of thebiologically active agent in question. Tubing made of other materialsmay be used in place of the fused silica tubing, or alternatively thelumen of the cannula can be coated with a substance to achievesubstantially the same result. The optimal material to be used may varydepending on the nature of the biologically active agent, and may bedetermined by experimentation.

The use of tubing with small internal diameter (ID), such as fusedsilica tubing with an ID of 100 μm, might be expected to reduce, ratherthan increase, recovery of sample due to the increased surface area tovolume ratio. Perhaps surprisingly, use of small ID fused silica tubingdoes not cause large losses of delivered products, for example, AAVvectors. Without intending to be limited by theory, the result may beexplained by the increased linear flow rate that results when a givendelivery rate (volume of product delivered per unit time) is maintainedconstant using smaller ID tubing. In addition, AAV appears to havelittle affinity for the surface of the fused silica tubing, which mayaccount for of the low losses.

The small ID of the fused silica tubing used in Example 1 has theadditional advantage of reducing the hold-up volume of the system. Forexample, a four-foot-long segment of fused silica tubing with an ID of100 μm has a lumen volume of less than 15 μl. Such low volumes reducesample consumption and significantly reduce waste of sample due to thehold-up volume of the delivery system. Reduced wastage of product isparticularly valuable when the biologically active agent is difficultand/or expensive to obtain, for example many recombinant proteins orgene therapy vectors.

FIG. 4 depicts an overview of an exemplary syringe pump that may be usedin combination with cannulas as described herein. Shown in FIG. 4 aresyringe saddle 60, syringe clamp 62, syringe clamp groove (retainer) 64,clutch lever 66, syringe driver 68, syringe plunger retainer 70, liquidcrystal display 72 and on/off switch 74. Syringe pumps useful in systemswith the cannulas described herein are commercially available, forexample under the name Medfusion 2010i (Medex, Inc., Carlsbad, Calif.).

FIG. 5 depicts an overview of a system including a cannula 70 asdescribed herein attached to a stereotactic frame 72. Cannula 70 mayalso be attached to syringe pump, for example via tubing 74.Stereotactic frames are commercially available, for example Lexellstereotactic frames (Ranfac Corp., Avon, Mass.).

In any of the systems described herein, the product contact portion maycomprise quartz silica (fused silica tubing in the cannula), USP classVII polypropylene (syringe and Luer hubs), cyanoacrylate (glue joints),and stainless steel (23G spacer).

Typically, the systems described herein are able to deliver product tothe brain with far less exposure to stainless steel than usingpreviously described systems, in which product is in contact withstainless steel along some or all of the entire length of the cannula.Reduced exposure of product to stainless steel, as provided in thedevices and systems of the present invention, reduces losses. Forexample, the cannula illustrated in FIG. 3B-6, as configured in thesystem illustrated in FIG. 1, has product contact surfaces comprisingalmost exclusively fused silica tubing and a USP Class VII polypropylenesyringe. The only other contact surface is the glue joint between theproximal end of the fused silica tubing and the Luer hub of the syringe,at which location product contacts cyanoacrylate adhesive and thecross-sectional surface of the proximal end of the stainless steelspacer. The exposure to steel in this system is minimal.

Cannulas of the present invention may also combine the step design andthe internal fused silica product contact surfaces to provide animproved cannula with reduced reflux, reduced surface-related losses ofagent and reduced hold-up volume.

Cannulas of the present invention may be sterilized using techniquesknown in the art including, for example, by standard ethylene oxide.Sterilized cannulas may optionally be individually packaged in a Tyvek®pouch.

Agents that can be delivered using a cannula of the present inventioninclude any material that may have a desired effect in the targettissue. For example, therapeutic drugs, proteins, plasmids or genetherapy vectors may be delivered into the brain of a subject.Non-therapeutic agents may also be added such a dyes, tracers, contrastagents and markers for imaging, diagnostic or research purposes.

For example, retroviral gene therapy systems have been described. See,e.g., U.S. Pat. No. 5,219,740; Miller and Rosman, BioTechniques (1989)7:980-990; Miller, A. D., Human Gene Therapy (1990) 1:5-14; Scarpa etal., Virology (1991) 180:849-852; Burns et al., Proc. Natl. Acad. Sci.USA (1993) 90:8033-8037; and Boris-Lawrie and Temin, Cur. Opin. Genet.Develop. (1993) 3:102-109. A number of adenovirus vectors have also beendescribed. See, e.g., U.S. Pat. Nos. 6,048,551, 6,306,652, Parks, R. J.,Clin. Genet. (2000) 58:1-11; Tsai et al., Curr. Opin. Mol. Ther. (2000)2:515-523.

Additionally, various adeno-associated virus (AAV) vector systems havebeen developed for gene delivery. AAV vectors can be readily constructedusing techniques well known in the art. See, e.g., U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92/01070(published 23 Jan. 1992) and WO 93/03769 (published 4 Mar. 1993);Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al.,Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B. J.Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. CurrentTopics in Microbiol. and Immunol. (1992) 158:97-129; Kotin, R. M. HumanGene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994)1:165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875.

Cannulas of the present invention can be used as part of aconvection-enhanced delivery (CED) system for administration to the CNS.For example, U.S. Pat. No. 6,309,634, the disclosure of which is herebyincorporated by reference in its entirety, describes methods of genetherapy in which agents are delivered to regions of the central nervoussystem by CED. Using CED, recombinant vectors can be delivered to manycells over large areas of the CNS. Moreover, the delivered vectorsefficiently express transgenes in CNS cells (e.g., glial cells).Cannulas of the present invention may be used with anyconvection-enhanced delivery device for delivery of recombinant vectors.In one embodiment, the device is an osmotic pump or an infusion pump.Both osmotic and infusion pumps are commercially available from avariety of suppliers, for example Alzet Corporation (Cupertino, Calif.),Hamilton Corporation, or Alza, Inc. (Palo Alto, Calif.).

Cannulas of the present invention can also be used for direct injectionor other methods of infusion, rather than CED.

Product may be delivered to a target tissue at a variety of flow rates,including but not limited to 0.2, 0.5, 0.7, 1.0, 1.5, 2.0, 3.0, 5.0, 10,15 or 20 μl/min. With reference to the embodiments illustrated in FIGS.1A and 2A, flow rates above 10-20 μl/min are difficult to achieve usingfour feet of fused silica tubing with 100 μm ID because of excessiveback-pressure at such high flow rates. This does not represent a seriouslimitation for convection enhanced delivery methods, however, which arepreferably performed at relatively low flow rates. Flow rates lower than0.2 μl/min may be difficult to achieve using the system illustrated inFIGS. 1A and 2A because the pump lacks a slow enough setting, but one ofskill in the art would be able to use a different pump and/or syringeconfiguration to achieve such low delivery rates.

The flow rate, and thus the pressure of the product as it is deliveredto the target tissue, may be increased, decreased, or held steadythroughout delivery. In a preferred embodiment, the flow rate is heldsubstantially constant throughout delivery, rather than being “rampedup” to a plateau.

Typically, a recombinant vector is delivered via CED devices as follows.An improved cannula of the present invention is inserted into CNS tissuein the chosen subject. Stereotactic maps and positioning devices areavailable, for example from ASI Instruments, Warren, Mich. Positioningmay also be conducted by using anatomical maps obtained by CT and/or MRIimaging to help guide the injection device to the chosen target.

Examples 2-5 disclose use of a cannula of the present invention todeliver the gene encoding hAADC to the brain of humans, rats andnon-human primates. Delivery of the hAADC gene may be helpful intreatment of Parkinson's disease (PD). PD is characterized in part bythe progressive loss of dopaminergic neurons in the substantia nigra anda severe decrease of dopamine in the putamen (Hornykiewicz (1975) Nat'lInst. Drug Abuse Res. Monogr. Ser. (3): 13-21). AADC is an enzyme in thedopamine biosynthetic pathway that converts L-dopa to dopamine. Previousstudies have shown that transfer of the cDNA encoding human AADC to rator non-human primate putamen can reduce effective L-dopa doses in animalmodels of PD, and thereby restore striatal dopamine to normal levels(Bankiewicz et al. (2000) Exp. Neurol. 164(1): 2-14; Sanchez-Pernaute etal. (2001) Mol. Ther. 4(4): 324-30). In human PD patients, this therapywould be expected to lower L-dopa requirements and extend the durationduring which clinical benefit of the drug is observed.

Example 2, along with associated tables and figures, provides a protocoland experimental results for delivery of a gene to the brain of aprimate using a cannula of the present invention (Clinical Device B).rAAV virions encoding hAADC (AAV-hAADC-2) are infused into the putamenof four normal rhesus monkeys and the distribution of AADC expression isdetermined by immunohistochemistry. Two infusion protocols are tested: aramped procedure (slow stepwise increases in rate from 0.2 μL/min to 1μL/min), and a non-ramped infusion at a constant rate of 1 μL/min. Theprimary endpoints are safety evaluation of the infusion procedures andassessment of transgene expression at 5.5 weeks post-infusion.

Clinical observations after vector infusions reveal no behavioralabnormalities during the study period. No differences in gross pathologywith either the ramped or non-ramped infusion procedure are observed.Histopathology is comparable in both groups, and reveals only minimallocalized inflammatory tissue reaction along the needle track inresponse to cannula placement and vector infusion. In addition, AADCimmunohistochemistry demonstrates that vector is distributed throughoutthe putamen, with no significant difference in volume of immunostainingwith either infusion procedure. Serum antibody levels against AAV2vector exhibited a minor increase after infusion.

The following examples are provided simply to illustrate a embodimentsof the present invention, and not to define or limit the invention.

EXAMPLE 1 Assembly and Packaging Procedures

A. Exemplary Cannula #1

An exemplary stepped cannula (such as shown in FIGS. 3A and 3B) wasproduced as follows.

Stainless steel tubing segments were cut in lengths and welded using aLasag Nd:YAG or Neodinium YAG (Yttrium aluminum garnet) laser, anultraviolet laser in the 454 nm wavelength region. The weld between the23G and the 19G segments was tested to be leak free and all weld jointswere tested to withstand a minimum pull force of 10 lbs. The weldbetween the 23G and 19G segments should be leak free to prevent anyproduct that may reflux up the outside of the needle from leaking intothe lumen of the cannula. For the same reason, the glue joint betweenthe exposed distal end of the fused silica tubing and the steel tubingportions of the cannula should be liquid-tight.

The needle was passivated and ultrasonically cleaned after laser stepsare completed. The fused silica tubing is cut and assembled to thestainless cannula with cyanoacrylate glue. A BD needle hub is attachedto the distal end of the tubing to finish the assembly. Prior topackaging, a plastic needle guard is placed over the proximal end of thecannula to protect the tip, then the entire assembly is packaged in apre-labeled Tyvek® pouch for sterilization.

The injection needle sub-assembly (INSA) was assembled by sliding asuccession segments of stainless steel tubing over a core segment 20 oftubing (9.67 inches long, 23 RW cutoff, 0.0250/0.0255 OD, 0.0125/0.0140ID, 0.006 wall). FIG. 3A. All dimensions relating to inner diameter(ID), outer diameter (OD) and tubing wall thickness (“wall”) areprovided in inches, with paired values X/Y representing minimum andmaximum tolerances.

Referring now to FIG. 3A, segment 22 (8.28 inches long, 19 RW cutoff,0.0415/0.0425 OD, 0.0255/0.0285 ID, 0.0075 wall) was placed over thecore segment 20 to leave a 0.390 inch (10 mm) of the core extendingbeyond the distal end of segment 22. Segment 24 (6.31 inches long, 17 RWcutoff, 0.0575/0.0585 OD, 0.0405/0.0435 ID, 0.008 wall) was placed overthe segments 20 and 22 to leave 1.970 inches of segment 22 extendingbeyond the distal end of segment 24. Segment 26 (6.31 inches long, 15 RWcutoff, 0.0715/0.0725 OD, 0.0595/0.0615 ID, 0.006 wall) was placed overthe segments 20, 22, 24 to leave 1.970 inches of segment 22 extendingbeyond the distal end of segment 26. Segment 28 (6.31 inches long,0.086/0.087 OD, 0.0735/0.0750 ID, 0.006 wall) was placed over thesegments 20, 22, 24, 26 to leave 1.970 inches of segment 22 extendingbeyond the distal end of segment 28. Segment 30 (1.58 inches long,0.108/0.110 OD, 0.0880/0.0895 ID, 0.010 wall) was placed over thesegments 20, 22, 24, 26, 28 to leave 7.090 inches of segment 20extending beyond the distal end of segment 30.

All components were laser welded in place. The distal weld seam betweensegments 20 and 22 was made 100% air tight, and the interior of segment1 was tested to ensure that there was no blockage (e.g. using a 0.012inch diameter wire or gage pin). All weld joints must withstand aminimum pull force of 10 pounds.

Once assembled, the INSA was passivated and ultrasonically cleaned asfollows: Oakite aluminum cleaned for 10 minutes, spray rinsed withdeionized water for 7 minutes, ultrasonically rinsed in alcohol, and airdried.

A needle guard 32 (9.0 inches long, 0.156 OD, 0.104/0.108 ID, 0.025wall) is placed over assembled segments 20, 22, 24, 26, 28, 30 to leavea 0.8 inch segment of segment 30 extending beyond the proximal end ofthe assembled segments.

INSAs were inspected to be free of traces of acid and cleaning solutionas follows: removed needle guard 32, soaked in alcohol bath, replacedneedle guard 32, blew air through distal end of needle guard 7,inspected liquid effluent at proximal end of segment 20, repeated untilall effluent appeared to be clean.

The distal end of the INSA was inspected to ensure that it was straight.

FIG. 3B illustrates the assembly of an exemplary injection needleassembly as described herein. As shown in FIG. 3B-1, the needle guard 32was removed from the INSA 35 as described above, and a length of fusedsilica tubing 40 was threaded through the core 23G tubing 15 of the NSA35, starting at the proximal end, until approximately 2 inches extendedbeyond the distal end of the NSA 35. Loctite® adhesive (Loctite® Prism®4011 adhesive, low viscosity) was applied to the exposed fused silicatubing 40, and then the fused silica tubing was withdrawn untilapproximately 1 inch remained beyond the distal end of the INSA whilespinning the INSA to evenly distribute adhesive. The bond strength ofthe adhesive bond is at least 5 pounds. The exposed fused silica tubingis trimmed so that 0.390 inches (10 mm) remained extending from thedistal end of the INSA, and the needle guard 32 was replaced.

A 48 inch length of FEP (Teflon) tubing ( 1/16 OD, 0.030 ID) wasprepared 31 and both ends were dipped in Loctite® primer (Loctite® 7701primer) and air dried. The proximal end of the fused silica tubing 40was threaded through the FEP tubing. Loctite® adhesive was applied tothe proximal end of the INSA. The distal end of the FEP tubing 31 wasquickly pushed over the needle end of the INSA. The bond strength of theadhesive bond is at least 4 pounds.

As shown in FIG. 3B-3, a 23G stainless steel spacer 47 (1 inch long, 23RW) was placed over the fused silica tubing 40. Loctite® adhesive wasapplied to the outside of the fused silica tubing 40 and to the outsideof the spacer 47, and the spacer 47 was inserted into the proximal endof the FEP tubing 31 until the proximal ends were flush. A 0.5 inch longsegment of PVC shrink tubing 49 (0.125 ID) was slipped over the proximalend of the FEP tubing 31.

As depicted in FIG. 3B-4, a 1/16 female Luer compression fitting 50 wasthen slipped over the proximal end of the FEP tubing 31 and a ferrule 51was placed approximately 1 inch over the proximal end of the FEP tubing31. Loctite® adhesive is applied to the outside of the FEP tubing 31 andthe ferrule 51 was pushed to place the proximal end of the ferrule flushwith the proximal end of the FEP tubing 31. Loctite® adhesive is appliedto seal the joints between the fused silica tubing 40, spacer 47, FEPtubing 31 and the ferrule 51.

The remaining fused silica tubing 40 extending proximally beyond theferrule 51 was scored and snapped off. As shown in FIG. 3B-5, theferrule 51 was then seated snuggly in the Luer compression fitting 50 (3pound minimum pull force) and the heat shrink tubing 49 was then heatshrinked over the joint between the proximal end of the INSA 35 and theFEP tubing 31.

The assembly was tested for air leaking, and a male Luer cap 55 wasadded to the compression fitting 50. (FIG. 3B-6).

The assembled INA may then be packaged and sealed in a Tyvek® pouch(4×23 inches) with a label, and placed in a labeled box for storage orshipment.

Although the tubing 31 was made of Teflon FEP, one of skill in the artwould recognized that any suitable tubing material could be used, or thetubing could be omitted altogether. The FEP tubing 31 was included asprotection for the fused silica tubing 40, and to help make sure thevery thin fused silica tubing was visible to operators of the system.Neither of these functions is essential. In addition, because the FEPtubing does not contact product tubing of other materials may be usedwithout regard to biocompatibility.

The finished cannula produced as described in herein comprises fivelayers of stainless steel tubing over 6.31 inches of its length (e.g.the length comprising tubing element 28), with an internal diameter of0.0125-0.0140 inches and an exterior diameter of 0.086 to 0.087 inches.This cannula has substantial rigidity along this segment, which preventsflexing of the cannula as it is inserted into the target tissue (e.g.the brain). In addition, a sixth layer of steel tubing 30 adds evengreater strength to the cannula over a 1.58 inch segment, which preventsthe cannula from being crushed or deformed when it is mounted in astereotactic frame during use, as illustrated in FIG. 5.

B. Exemplary Cannula #2

Another cannula was produced similar to the one described in Example 1A.As shown in FIG. 6, the cannula 80 is composed of four layers of 304surgical steel fused together by laser welding in a step design, endingin 30 gauge tubing. The steel cannula (approximately 24.6 cm from end toend, including needle tip) is lined with fused silica of 100 μm innerdiameter 82 which also forms the tip of the delivery device by extending1 cm beyond the steel. Approximately 1.2 meters (122 cm) of additionalfused silica 82 covered with Teflon tubing 84 connect to a Luer hub 86.A 1-inch 30 gauge steel spacer 88 between the fused silica and Teflontubing is sealed and attached to the Luer hub with medical gradecyanoacrylate glue.

EXAMPLE 2 Delivery of Recombinant Viral Vectors Encoding AADC to PrimateBrain

Recombinant AAV vector encoding human AADC (AAV-hAADC-2) was preparedand delivered to the putamen of rhesus monkeys as follows.

Recombinant Vector Production

Recombinant AAV2 was generated by a triple transfection protocol(Matsushita et al. (1998) Gene Ther. 5(7): 938-45). Briefly, afterexpansion of cells from the HEK 293 working cell bank through a seriesof disposable culture ware in DMEM containing 10% fetal bovine serum and2 mM glutamine, cells were co-transfected with three plasmids(pAAV-hAADC-2, pHLP 19 and pladeno5). The rAAV-hAADC-2 vector clone isthe same as that described previously (Sanftner et al. (2004) Mol. Ther.9(3): 403-9). Plasmids pHLP 19 and pladeno5 are described more fully atU.S. Pat. Nos. 5,139,941; 5,622,856; 6,001,650 and 6,004,797, thedisclosures of which are hereby incorporated by reference in theirentireties.

After an appropriate transfection time, the medium containing thetransfection reagent was replaced with serum-free medium and the cellswere incubated further to allow vector production. Cells were harvested,concentrated by centrifugation, and lysed by a freeze/thaw method torelease the AAV-hAADC-2 vector. After centrifugation to remove cellulardebris, the lysate was treated with Benzonase®, calcium chloride, andprecipitated with polyethylene glycol. Vector was purified by two cyclesof isopycnic gradient ultracentrifugation in cesium chloride.AAV-hAADC-2 was concentrated, and diafiltered with sterile, bufferedsaline (PBS) containing 5% sorbitol. Poloxamer 188™ (0.001%) was added,the material is sterile filtered (0.22 μm), and stored frozen at −70° C.Vector purity was assessed by SDS-PAGE. Purified rAAV2 vector used inthis study showed only VP1, VP2, and VP3 by silver staining of SDS-PAGEgels. Titer was determined by real-time Q-PCR analysis of vectorgenomes.

Surgical Procedures

Magnetic resonance imaging (MRI) was performed on each monkey prior tosurgery to identify stereotaxic coordinates (based on the anatomicalstructure of the putamen). Two sites were targeted in each hemispherewith one site centered in the rostral putamen and a second in the caudalputamen. Adult rhesus monkeys (n=4) were immobilized with a mixture ofketamine (Ketaset®, 10 mg/kg, intramuscular injection) and Valium® (0.5mg/kg, intravenous injection), intubated and prepared for surgery.Isotonic fluids were delivered intravenously at 2 mL/kg/hr. Anesthesiawas induced with isoflurane (Aerane®, Omeda PPD, Inc., Liberty, N.J.) at5% v/v, and then maintained at 1%-3% v/v for the duration of thesurgery. The animal's head was placed in an MRI-compatible stereotaxicframe. Core temperature was maintained with a circulating water blanketwhile electrocardiogram, heart rate, oxygen saturation and bodytemperature are continuously monitored during the procedure. Burr-holeswere made in the skull with a dental drill to expose areas of the durajust above the target sites. AAV-hAADC-2 was infused by CED (Liebermanet al. (1995) J. Neurosurg. 82(6): 1021-9; Bankiewicz et al. (2000) Exp.Neurol. 164(1): 2-14). Each monkey received a total of 3×10¹¹ vg in 200μL spread over four sites (50 μL per site with two sites perhemisphere). Infusion cannulae were manually guided to the putamen ineach brain hemisphere, and the animals received bilateral infusions(i.e. sequential infusions to the rostral and caudal sites within bothhemispheres) of AAV-hAADC-2 (1.5×10¹² vg/mL) at infusion rates of 0.2μL/min (10 min), 0.5 μL/min (10 min), 0.8 μL/min (10 min) and 1 μL/min(35 min) for the left hemisphere and a constant rate of 1 μL/min (50min) for the right hemisphere. Actual stereotaxic coordinates for eachanimal were: MR15101M rostral putamen AP: 18, ML: ±10.5, DV: 20, caudalputamen AP: 15, ML: ±13, DV: 20, R211101M rostral putamen AP: 24, ML:±12.5, DV: 20, caudal putamen AP: 21, ML: ±13.5, DV: 20, MR15109Mrostral putamen AP: 12, ML: ±13, DV: 20, caudal putamen AP: 15, ML: ±12,DV: 20, R23700M rostral putamen AP: 21, ML: ±13.5, DV: 21, caudalputamen AP: 24, ML: ±12.5, DV: 20. Approximately 10 minutes afterinfusion, the cannulae were removed, the wound sites were closed, andthe monkey was monitored for recovery from anesthesia and then returnedto its home cage for continuing observations.

Histology and Immunohistochemistry

For histological studies, animals were perfused via intracardiac salineinfusion followed by 10% neutral buffered formalin (NBF). The brainswere then removed and sliced in a brain mold into coronal blocks (8-10mm). Harvested brain blocks were fixed by immersion in 10% NBF fixative.The tissue blocks were transferred 2-3 days after fixation intoascending concentrations of PBS/sucrose solution (10, 20 and 30%) over a3-5 day period. Brains were frozen in a bath of isopentane, cooled ondry ice and cut serially into 40 μm thick coronal sections on acryostat. Every tenth section was stained with Hematoxylin and Eosin(H&E) solutions (Richard Allen Scientific, Kalamazoo, Mich.) forhistopathological analysis. Immunohistochemistry was carried out onfree-floating sections with a primary antibody specific for AADC(Chemicon, Temecula, Calif., 1:1,500). Sections were incubated in 3%hydrogen peroxide for 30 min to quench endogenous peroxidases. Afterblocking for non-specific binding with 10% normal goat serum, sectionswere incubated in primary antibody overnight at room temperature, thenwith a biotinylated anti-rabbit IgG antibody (Vector Laboratories,Burlingame, Calif., 1:300) with streptavidin-conjugated horseradishperoxidase (Vector Laboratories, 1:300) at room temperature, both for 1h. The complex was visualized with 3-3′-diaminobenzidine (DAB, VectorLaboratories) and hydrogen peroxide. Sections were mounted on SuperfrostPlus® slides (Brain Research Laboratories, Newton, Mass.), dried,dehydrated in ascending ethanol series, cleared in xylene, and mountedwith Cytoseal-XYL (Richard-Allen Scientific, Kalamazoo, Mich.).Anterior-to-posterior distribution of hAADC immunostaining wasdetermined by the formula (n×10×40 μm) where n is the number of sectionswith hAADC-positive cells, 40 μm is the thickness of the section, andevery tenth section was examined. The volume of distribution wasestimated in serial sections (every tenth), stained for AADC with theOptical Fractionator-Optical Dissector design-based stereology methodunder 63× magnification on a Zeiss microscope equipped with a videocamera and Stereoinvestigator™ stereology software (Microbrightfield,Williston, Vt.). CEE is <5% for each group. Results are reported asmean±SD. Student's t-test was used to measure statistical significance.

Real-Time Quantitative PCR

The vector AAV-hAADC-2 used in this study contains the human AADC targetcDNA. The real-time Q-PCR primers and probe anneal to exons 2 and 3 ofthe AADC gene, spanning an intron not present in the vector sequence,thereby minimizing amplification of genomic DNA. Real-time Q-PCR isstandardized with linearized plasmid DNA containing the vector insertand vector genomes were quantified as described previously (Sommer etal. (2003) Mol. Ther. 7(1): 122-8).

Neutralizing AAV Antibody Titering

The neutralizing antibody (NAb) titer of serum or plasma was determinedin vitro in a cell-based assay. A defined number of AAV2 vectorparticles encoding a β-galactosidase reporter gene (AAV2-LacZ) wereincubated with test serum for 1 h at 37° C. before addition of themixture to HEK-293 cells near confluence in 96-well plates. Control(100%) AAV2 transduction was defined as the amount of β-galactosidaseactivity measured in culture 24 h after transduction with AAV2-LacZ inthe presence of naive mouse serum (NMS). A half-log serial dilution ofthe test serum in NMS was made to determine the highest dilution of testserum that results in 50% or greater inhibition of β-galactosidaseexpression. Each dilution series was tested in triplicate. A referenceplasma with a well-defined AAV2 neutralizing titer was run in each assayand a negative control (NMS only) was used to determine the assaybackground. The titer of NAb was defined as the two dilutions thatbracket the 50% inhibition level, e.g. 1:100 to 1:316.

Bridging ELISA

Titer plates (96 well) were coated with AAV2 particles and thenincubated with test sample (serum or plasma). Plates were rinsed andthen incubated with biotinylated AAV2 particles, which were thendetected with HRP-conjugated Streptavidin. The biotinylated AAV2particles can only be captured by multivalent antibodies forming abridge between two AAV2 particles. A very low non-specific backgroundsignal in this assay permitted testing of undiluted or low dilutions oftest articles, and the assay has higher sensitivity than a classicalELISA, in which primary antibody in the test sample is detected by anenzyme-conjugated secondary antibody. The bridging assay allows directtiter comparisons between different species and classes of antibodies.The assay was standardized with known amounts of purified mousemonoclonal antibody “A20” that recognizes AAV2 (Grimm et al. 1998). Thequantification limit of this assay was approximately 15 ng/mL anti-AAV2antibody. Human samples with a NAb titer of 1:100 contained between 1and 10 μg/mL of antibody equivalent to A20. The average inter-assayvariability for 65 human samples that underwent replicate testing bythis assay was 23%.

Experimental Design

Recombinant AAV2 vectors transduce brain tissue efficiently, buttransduction levels decline significantly in the presence of highneutralizing antibodies (NAb) titers (>1:1200) (Sanftner et al. (2004)Mol. Ther. 9(3): 403-9). Therefore, four male rhesus monkeys with NAbtiters of ≦1:100 were selected for AAV2 infusions (Table 1). MRI scanswere performed prior to AAV2 delivery to determine stereotaxiccoordinates for vector administration. Animals were bilaterally infusedwith 1.5×10¹¹ vg of AAV-hAADC-2 in two 50 μL infusions (7.5×10¹⁰vg/site) in each hemisphere (3.0×10¹¹ vg/brain). Ascending infusionrates (ramp) of 0.2 μL/min (10 min), 0.5 μL/min (10 min.), 0.8 μL/min(10 min) and 1 μL/min (35 min) were used for the left hemispheres,whereas a constant rate of 1 μL/min for 50 min (non-ramp) was used forthe right hemispheres. Animals were monitored for 5.5 weeks, a time spansatisfactory for hAADC expression to become relatively stable. Primaryendpoints included AADC expression as determined by immunohistochemistryand safety assessments as determined by clinical observations andhistopathology. In addition, serum samples, collected at baseline and atthe end of the study, were tested for the presence of both neutralizingand total antibodies against AAV.

TABLE 1 ANTI-AAV SERUM ANTIBODY (NAB) TITERS AND BRIDGING ELISA DATANon-human Bridging ELISA (μg/mL Primate ID Sample NAb Titer anti-AAV Ab)MR15102M Pre-treatment  1:1-1:3.1  0.036 1:3.1-1:10 Post-treatment 1:1-1:3.1 0.24 ± 0.08 1:3.1-1:10 MR15109M Pre-treatment 1:3.1-1:10Below Detection 1:3.1-1:10 (<0.015) Post-treatment 1:3.1-1:10 0.43 ±0.35 1:3.1-1:10 R211101M Pre-treatment  1:1-1:3.1 0.11  1:1-1:3.1Post-treatment  1:31-1:100 0.63 ± 0.07  1:10-1:31 R23700M Pre-treatment 1:10-1:31 0.24  1:31-1:100 Post-treatment  1:31-1:100 1.3 ± 0.7 1:100-1:316

Infusion Device Development and Vector Recovery

A prototype infusion device for human use (“Clinical Device A,” or CDA)was composed of a 25-cm stainless steel cannula, made to fit a standardLeksell® stereotaxic frame. The CDA cannula was composed of four steppedlayers of medical grade stainless steel tubing to provide rigidity andminimize internal hold-up volume. The steel CDA cannula was connected toa syringe via 1.2 meters of Teflon® tubing. Vector recovery studies atflow rates up to 1 μL/min reveal that 90% of the vector product wasadsorbing to the device (Table 2), despite the 0.01% Poloxamer 188included as a surfactant in the product formulation. A 1-hr flush of thedevice with vector improves subsequent recovery, but vector loss wasstill approximately 40%. Further testing for vector absorption includedtesting of stepped stainless steel cannulas in which the productcontacts different tubing materials at flow rates of ≦1 μL/min. (Example1A and 1B). Excellent vector recovery was observed for cannulascomprising fused silica, Tygon®, and silicone tubing in contact with theAAV vector. Other materials such as steel, Teflon (PTFE and FEP) andpolyimide bound significant amounts of vector.

TABLE 2 VECTOR RECOVERY: PRECLINICAL, CLINICAL DEVICE A AND CLINICALDEVICE B Preclinical Device Clinical Device A Clinical Device B Productcontact Fused silica, Teflon ®, No. 304 stainless steel, Fused silica,surfaces polypropylene (Luer Teflon ®, polypropylene (Luer couplings)polypropylene (Luer and syringe) lock and syringe) Internal hold-upvariable 350 μL 12 μL volume Vector recovery after 63 ± 16% 9 ± 4% 101 ±6% ≦50 μL flush volume (±SD) Vector recovery after not done 60 ± 15% notdone 500 μL of flush at 8 μl/min (±SD)

Much of the vector loss was observed only at low flow rates. Forexample, in Teflon tubing, vector loss was inversely proportional to thelinear flow rate. Ninety percent, of the vector was lost at 1 μl/min (4mm/min through 1.2 meters of tubing), whereas acceptable vector recovery(>80%) could be attained in the same tubing at flow rates above 100μL/min. In order to maximize the linear flow rate and to eliminate allcontact of vector with Teflon and steel surfaces, the entire core of theclinical device was lined with fused silica of inner diameter 100 μm(Example 1B, FIG. 6). In this device (“Clinical Device B”, or CDB), thesteel cannula surrounds the fused silica to provide rigidity, and thefused silica extends 10 mm beyond the tip of the steel cannula (FIG. 6).Two external steps near the needle tip are included to minimizepotential reflux along the needle track. An additional 1.2 meters offused silica connects the CDB cannula to a Luer hub and is covered byTeflon tubing only to provide protection. The CDB was manufactured andassembled in accordance with cGMP and terminally sterilized by ethyleneoxide gas.

Quantitative recovery of vector was evaluated through mock infusionswith preclinical and clinical devices. For the preclinical device, 400μL of vector solution was drawn from the distal end into a length ofTeflon tubing that was then coupled to a 7 cm cannula composed of fusedsilica surrounded by a 4 cm piece of 27-gauge steel tubing. Afterfilling the cannula at 100 μL/min, an additional 20 μL flush wasdispensed before collecting vector for recovery assays. Four sampleswere collected from two devices at flow rates from 0.2 to 1.0 μL/min(ramped procedure) with a programmable syringe pump.

As shown in Table 2, the average vector recovery from the preclinicaldevices under these conditions was 63±16% (±SD). For the clinicaldevices, AAV-hAADC-2 vector was diluted to 5×10¹¹ vg/mL, loaded intosyringes and attached to the devices. After fill, Clinical Device A wasflushed with 500 μL of vector solution at 8 μL/min (62.5 min), whileClinical Device B was flushed with a total of 50 μL of vector solutionat 4 μL/min (12.5 min). Two sequential aliquots of 50 μL were collectedfrom three sets of each device at flow rates from 0.2 to 1.0 μL/min.Vector concentration in each sample was determined by real-timequantitative PCR (Q-PCR).

Recovery for Clinical Device A was only 60±15% after the extensiveone-hour flush, whereas complete recovery of vector (101±6%) wasobserved for Clinical Device B. Potency of vector samples recovered fromClinical Device B was confirmed by determining the infectious titer(see, Zhen et al. (2004) Hum. Gene Ther. 15(7):709-715. No significantdecrease in specific activity (infectious units/vg) was observed.

Immunohistochemistry and Quantitation of hAADC Expression In-Vivo

Immunohistochemical analysis of hAADC expression was performed on eachbrain hemisphere at 5.5 weeks post-AAV-hAADC-2 infusion to determine ifthe vector distribution was different after ramped vs. non-rampedinfusion with Clinical Device B. All monkeys exhibited hAADC expressionwithin the putamen. Serial sections were examined with brightfieldmicroscopy for hAADC-positive cells. The volume of distribution andAnterior-Posterior (A-P) spread of hAADC transgene-positive cells weredetermined for all animals.

FIG. 7 shows immunohistochemical staining for the hAADC transgene incross-sections through the infusion site. Images are of whole mounts ofsections from animals MR15102M (A), MR15109M (B), R23700M (C) andR211101M (D). Sections are oriented from a caudal view with the righthemisphere on the right side of the image and the left hemisphere on theleft side of the image. In all animals, transgene expression waslocalized to the putamen. No hAADC expression was detected in corticalregions except in direct line with the infusion track as illustrated inFIG. 7B. No difference in the number of AADC-positive cells or intensityof hAADC staining was seen in a comparison of the right and lefthemispheres.

A higher magnification image of the infusion site of the putamen in arepresentative animal from the left hemisphere that received rampedinfusion (FIG. 8A), or the right hemisphere that received non-rampedinfusion (FIG. 8B) illustrates the hAADC transgene expression in mediumspiny neurons. Immunohistochemical staining for hAADC expression wasseen in all (8/8) of the infused hemispheres. AAV-hAADC-2 administrationresulted in good expression and coverage of the putamen with a similardistribution of AAV-hAADC-2 with either the ramped (left hemisphere) ornon-ramped (right hemisphere) infusion procedure.

Quantitation of the estimated volume of hAADC distribution in serialsections stained with anti-hAADC antibody was performed withStereoinvestigator™ stereology software (Microbrightfield, Williston,Vt.). The Anterior-Posterior (A-P) distribution, a one dimensionalmeasure of distribution from rostral to caudal, and volume of hAADCimmunostaining were determined separately for each hemisphere of thefour AAV-treated non-human primates (Table 3). The mean A-P distributionand the mean volume for either the right or left hemisphere were eachbased on four hemispheres. The mean A-P distribution for the lefthemisphere (ramped delivery) was 9,600 μm±2,422 μm (SD) and the meanvolume was 238 mm³±121 mm³. The mean A-P distribution for the righthemisphere (non-ramped delivery) was 9,606 μm±2,037 μm and the meanvolume was 284 mm³±55 mm³. There was no significant difference in meanvolume or mean A-P distribution between ramped or non-ramped by anunpaired Student's t-test (P=0.9973 for A-P distribution comparison andP=0.5187 for spread volume comparison). The non-ramped infusion did notresult in reflux of vector along the cannula track or a decrease intransgene-derived hAADC distribution. The lack of reflux may also inpart be due to the multiple step design of the cannula.

TABLE 3 ANTERIOR-TO-POSTERIOR (A-P) DISTANCE OF SPREAD AND SPREAD VOLUMEOF AADC IN NON- HUMAN PRIMATES INFUSED WITH AAV-hAADC-2 A-P DistributionSpread volume (μm) (mm³) Right (non-ramped infusion) Animal I.D. MR151098,822 272.1 R23700M 12,400 346.5 R211101M 9,600 301.5 MR15102M 7,600214.4 Mean right hemisphere 9,606 283.6 (non-ramped infusion) StandardDeviation 2,037 55.4 Left (ramped infusion) Animal I.D MR15109 8,400110.2 R23700M 12,800 402.3 R211101M 10,000 217.3 MR15102M 7,200 222.2Mean left hemisphere 9,600 238.0 (ramped infusion) Standard Deviation2,422 121.1

Histopathology

Histopathological analysis of serial sections stained with H&E wasperformed on all animals to determine the effect of cannula placementand AAV-hAADC-2 infusion with either ramped or non-ramped delivery. FIG.9 shows H&E stained sections within the putamen from a representativeanimal, R211101M, at 5× magnification. Animal R211101M receivedbilateral CED of AAV-hAADC-2 by the non-ramped infusion procedure in theright hemisphere (Panel A) and the ramped infusion procedure in the lefthemisphere (Panel B). Images illustrate the area adjacent to the cannulatrack at the mid-caudal putamen level. All H&E stained slides werereviewed by a neuropathologist (Pathology Associates Inc.), blinded totreatment conditions. Some mononuclear cellular infiltration was seen inthe putamen with mild perivascular cuffing. Both putamina contained afew infiltrated blood vessels and mild parenchymal infiltration.Histopathologic appearance of the right and left hemispheres wassimilar, with slight inflammatory tissue reaction at the infusion site.

Development of Neutralizing Antibodies

Neutralizing antibody (NAb) and total antibody titers to AAV capsid weredetermined for serum samples collected prior to infusion of vector, andat the time of necropsy. Slight rises in anti-AAV antibody levels weredetected by bridging ELISA in all animals after bilateral infusion ofAAV-hAADC-2 (Table 1). The results for two NAb assays are shown inTable 1. The bridging ELISA is standardized with anti-AAV2 mousemonoclonal antibody. The average of two results is shown forpost-treatment samples and a single result is shown for pre-treatmentsamples. The animal (R23700M) with the highest serum neutralizingantibody titer (1:10 to 1:100) before treatment has a post-treatmentantibody increase to 1:31-1:316 on Day 42. This animal had similar hAADCtransgene distribution when compared to the other animals and thus therewas no apparent inhibition of vector spread associated with the highertiter.

Clinical Observations

Monkeys were evaluated daily for clinical signs, food consumption, andbody weight. Post-surgical daily clinical observations indicated thatthe animals tolerated the CED procedure well and did not displaybehavioral changes. There were no AAV-hAADC-2 treatment-related clinicalsigns or changes in body weight. Observations made during thepost-treatment period were similar to those commonly observed inlaboratory-housed rhesus monkeys that undergo similar surgicalprocedures.

Results

An embodiment of the cannula of the present invention was tested toassess its ability to effectively deliver rAAV vector to primate brain,which may serve as a model for delivery of therapeutic rAAV vectors fortreatment of Parkinson's disease in a human subject. Mock infusionsdesigned to test vector delivery established that essentially 100% ofthe intended dose can be delivered with a cannula as described herein,preferably avoiding contact of vector with Teflon or steel surfaces.

Stereotaxic administration of AAV-hAADC-2 into the putamen of fournon-human primates was performed by comparison of a ramped (gradedincrease in infusion rate) vs. a non-ramped (constant rate) infusionprocedure. Expression of hAADC was detectable by immunohistochemistryand was distributed broadly in the putamen. Stereological quantitationof the volume of transgene-derived hAADC demonstrated similardistribution in hemispheres receiving either infusion procedure.Furthermore, the constant flow rate did not result in excessive vectordeposits along the needle track. Histopathologic analysis revealed onlyslight tissue inflammatory reaction localized to the area of the cannulainsertion track, suggesting no safety concerns. There was no apparentdifference in the degree of cellular infiltration or inflammationbetween the left and right putamen (i.e. ramped vs. non-rampedinfusion). No abnormal clinical observations were seen after surgery andintraputamenal infusion in any animals.

In addition to device and infusion parameters, another importantconsideration for effective AAV-mediated gene delivery into anycompartment is potential neutralization by anti-AAV antibodies. There isa broad range of pre-existing AAV neutralizing antibody titers in humans(Blacklow et al. (1968) J. Natl. Cancer Inst. 40(2): 319-27) that havethe potential of adversely affecting the efficacy of gene therapytechniques. Any AAV-mediated gene therapy approach must anticipate suchhurdles.

For example, in a model system utilizing SCID mice wherein humanAAV2-neutralizing antibody titers could be established at variouslevels, it was observed that titers<1:10 significantly impacted livertransduction of AAV-Factor IX after intravenous administration (Scallanet al. (2004) American Society of Gene Therapy, Minneapolis, Minn.,Abstract #753 S286). Delivery of AAV2 to the putamen was assumed to beless subject to neutralization by circulating antibodies due to theimmune-privileged status of the CNS. In fact, studies performed in ratspre-immunized systemically with AAV2 and then infused intrastriatallyconfirmed significant protection from neutralization with a decrease intransduction observed only when Nab titers exceeded 1:1200 (Sanftner etal. (2004) Mol. Ther. 9(3): 403-9).

Experiments described herein utilized animals with pre-existing NAbtiters ranging from 1:1 to 1:100 in order to exclude neutralizingantibodies as a confounding variable, and these titers have no apparentimpact on hAADC expression in putamen. Moreover, post-infusion titersrose only slightly after vector administration, thereby affirmingwell-targeted and minimally-disruptive gene delivery with the currentdevice and infusion conditions. These results also suggest that repeatintrastriatal infusions of AAV2 may be feasible in human patients.

In summary, non-ramped infusion of AAV-hAADC-2 to monkey putamen via aninfusion device of the present invention (Clinical Device B) was welltolerated. Transgene (hAADC) expression and distribution in the putamenwere comparable to more complicated and time-consuming ramp-up flowconditions. Given that gene therapy of neurodegenerative diseases andother CNS disorders is an expanding field (Tinsley and Eriksson (2004)Acta Neurol. Scand. 109(1): 1-8), the present results suggest that thedesign of Clinical Device B represents an important advancement inmethodology for this field. The device and infusion parameters of thepresent invention are likely to be applicable for striatal delivery ofAAV2 in PD patients, and also for targeting different anatomic sites,delivering a variety of therapeutic drugs or gene therapy agents, andtreating an assortment of CNS clinical indications.

Examples are intended to illustrate the invention and do not by theirdetails limit the scope of the claims of the invention. While preferredillustrative embodiments of the present invention are described, it willbe apparent to one skilled in the art that various changes andmodifications may be made therein without departing from the invention,and it is intended in the appended claims to cover all such changes andmodifications that fall within the true spirit and scope of theinvention.

All publications, patents, and patent application publications, andreferred to herein are hereby incorporated by reference in thereentireties.

1. A stepped cannula configured for delivering one or more materials tothe brain, comprising: (a) two or more co-axially disposed segments,each segment having an exterior diameter that defines the exteriordiameter of the cannula, wherein the exterior diameter of the segmentsis different; and (b) one or more tubular components extending throughthe lumen of the cannula, wherein at least one tubular componentcomprises a tubing that reduces loss of material delivered to the brainthrough the cannula, and further wherein the at least one tubularcomponent extends from 1 mm to 10 mm beyond the distal end of the lumen.2. The cannula of claim 1, wherein the tubing that reduces loss ofmaterial is a fused silica tubing.
 3. The cannula of claim 1, whereinthe cannula comprises two, three, four, five or six co-axially disposedsegments.
 4. The cannula of claim 1, wherein the exterior diameter ofthe segments decreases from the proximal end to the distal end of thecannula.
 5. The cannula of claim 1, wherein the cannula has a constantinterior diameter.
 6. The cannula of claim 2, wherein the cannulafurther comprises FEP tubing disposed around the fused silica tubing. 7.The cannula of claim 1, wherein the two or more co-axially disposedsegments comprise stainless steel.
 8. The cannula of claim 7, whereinthe lumen of one or more of the stainless steel segments is coated withone or more polymers.
 9. The cannula of claim 1, wherein the cannulacomprises five stainless steel segments.
 10. The cannula of claim 1,wherein the lumen of the cannula is operably connected to a reservoircomprising the one or more materials to be delivered through thecannula.
 11. The cannula of claim 10, wherein the reservoir comprises asyringe.
 12. The cannula of claim 11, wherein the syringe is operablylinked to a pump.
 13. The cannula of claim 12, wherein the pump isprogrammable.
 14. The cannula of claim 12, wherein the pump is aconvection enhanced delivery pump.
 15. The cannula of claim 10, whereinthe reservoir is operably linked to the one or more tubing componentsextending through the lumen of the cannula.
 16. The cannula of claim 1,wherein the at least one tubular component extends from 1 mm to 5 mmbeyond the distal end of the lumen.
 17. The cannula of claim 1, whereinthe cannula is operably linked to a pump.
 18. The cannula of claim 17,wherein the pump is programmable.
 19. The cannula of claim 17, whereinthe pump is a convection enhanced delivery pump.